Epstein-Barr virus (EBV) is an extremely successful pathogen, being able to establish a lifelong latent infection within B lymphocytes of its human host, with overt disease in healthy individuals restricted to a self- limiting mononucleosis in ~40% of individuals when infection is delayed until the second decade of life. However, a breakdown in cellular immunity, particularly as a consequence of AIDS, remains a significant risk factor for development of EBV-associated lymphoma and lymphoproliferative disease, underscoring the highly evolved equilibrium that exists between EBV and its host. Establishment of this equilibrium is dependent initially on a highly coordinated and controlled expression of the nine EBV latency-associated proteins and a subset of the viral miRNAs - the latency III (Lat III) program. The long-term objective of the research proposed in this application is to fully understand the mechanisms that contribute to EBV persistence through regulating the Lat III program, i.e., during the early establishment phase of persistent infection. Central to this phase are a family of six EBV nuclear proteins (EBNAs) that are expressed from one of two common promoters (Wp and Cp). The EBNAs primarily function as transcription factors (EBNA1 is also the viral genome-maintenance protein) to promote virus persistence through their regulation of cellular and viral latency-gene expression. The foundation for this application are our recent discoveries of two novel and apparent posttranscriptional regulatory mechanisms of controlling EBNA expression, and the discovery of a novel family of latency- associated EBV RNAs that may contribute to Lat III through a potential regulatory function and/or novel protein expression. Under Aim 1, we propose to identify the EBV gene responsible for the trans-repression of protein expression dependent on the EBNA promoter Wp, and ultimately its mechanism of action and importance to EBV biology and persistence. In Aim 2 we propose to define the contribution of the EBV BHLF1 gene locus of the virus genome to latent infection, expanding on preliminary data that suggests BHLF1 may function during latent infection as long noncoding RNA (lncRNA) important for the proper splicing of the EBNA mRNAs. Finally, in Aim 3 we will characterize and explore the contribution to EBV latency of a family of novel RNA transcripts that span the EBNA promoter Cp.